The aim of this study is to explore the mechanism of ligustilide, the main active constituent of essential oils of traditional Chinese medicine Angelicae Sinensis Radix, on alleviating oxygen-glucose deprivation/reperfusion(OGD/R) injury in PC12 cells from the perspective of ferroptosis. OGD/R was induced in vitro, and 12 h after ligustilide addition during reperfusion, cell viability was detected by cell counting kit-8(CCK-8) assay. DCFH-DA staining was used to detect the level of intracellular reactive oxygen species(ROS). Western blot was employed to detect the expression of ferroptosis-related proteins, glutathione peroxidase 4(GPX4), transferrin receptor 1(TFR1), and solute carrier family 7 member 11(SLC7A11), and ferritinophagy-related proteins, nuclear receptor coactivator 4(NCOA4), ferritin heavy chain 1(FTH1), and microtubule-associated protein 1 light chain 3(LC3). The fluorescence intensity of LC3 protein was analyzed by immunofluorescence staining. The content of glutathione(GSH), malondialdehyde(MDA), and Fe was detected by chemiluminescent immunoassay. The effect of ligustilide on ferroptosis was observed by overexpression of NCOA4 gene. The results showed that ligustilide increased the viability of PC12 cells damaged by OGD/R, inhibited the release of ROS, reduced the content of Fe and MDA and the expression of TFR1, NCOA4, and LC3, and improved the content of GSH and the expression of GPX4, SLC7A11, and FTH1 compared with OGD/R group. After overexpression of the key protein NCOA4 in ferritinophagy, the inhibitory effect of ligustilide on ferroptosis was partially reversed, indicating that ligustilide may alleviate OGD/R injury of PC12 cells by blocking ferritinophagy and then inhibiting ferroptosis. The mechanism by which ligustilide reduced OGD/R injury in PC12 cells is that it suppressed the ferroptosis involved in ferritinophagy.
Animals ; Rats ; PC12 Cells ; Ferroptosis/genetics* ; Reactive Oxygen Species ; Transcription Factors ; Glutathione

[Abstract] Objective To investigate the effect and possible mechanism of cell cycle-dependent kinase (Cdk)5 inhibitor Roscovitine on 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced pathological changes in brain regions associated with Parkinson’ s disease (PD) model mice. Methods The effect of Roscovitine on the relative expression levels of P25 and Cdk5 proteins was detected by Western blotting in MPP

Animals ; Apoptosis ; Brain/metabolism* ; Male ; Phosphatidylinositol 3-Kinases/metabolism* ; Rats ; Rats, Sprague-Dawley ; Subarachnoid Hemorrhage/drug therapy* ; Thyroxine/pharmacology*

Objective: To explore the role and related mechanism of mammalian sterile 20-like kinase 1(Mst-1)in regulating hypoxia reoxygenation (HR) induced myocardial cell autophagy and apoptosis. Methods: Enzyme digestion method combined with differential adherent method was used to culture neonatal mouse myocardial cells. HR model was established by hypoxia for 24 hours and reoxygenation for 6 hours. The experimental groups including control group (normal cultured cardiomyocytes), Mst-1 empty virus group (cardiomyocytes transfected with recombinant lentiviral empty vector for 48 hours), Mst-1 knockdown group (recombinant lentivirus carrying Mst-1small interfering RNA (siRNA) was transfected into cardiomyocytes for 48 hours), Mst-1 overexpression group (cardiomyocytes were transfected with recombinant lentivirus carrying Mst-1 gene for 48 hours), HR group (cardiomyocytes exposed to HR), Mst-1 knockdown+HR group (HR model of cardiomyocyte was established 48 hours after transfection with recombinant lentivirus carrying Mst-1siRNA) and Mst-1 overexpression+HR group (HR model of cardiomyocyte was established 48 hours after transfection with recombinant lentivirus carrying Mst-1 gene). Real-time fluorescence quantitative RCR (qPCR) and Western blot were used to detect the relative expression of Mst-1 mRNA and protein in the cells, immunofluorescence staining was used to detect cardiomyocyte troponin T (cTnT), and autophagosomes and autophagy enzyme changes. TUNEL method was used to detect myocardial cell apoptosis, Western blot was adopted to detect autophagy-related protein microtubule-related protein 1 light chain 3 (LC3) Ⅱ/LC3 Ⅰ, P62 and apoptosis-related protein cleaved-caspase 9, pro-caspase 9, cleaved-caspase-3, pro-caspase-3, and myeloid leukemia 1 (MCL-1) expression. MCL-1 inhibitor A1210477 was used to validate the signaling pathway of Mst-1 on regulating cardiomyocyte apoptosis and autophagy. Results: Immunofluorescence detection revealed that the cultured cells expressed cardiomyocyte-specific marker cTnT. The expression of Mst-1 in cardiomyocytes increased in HR model. Lentiviral transfection could effectively inhibit or overexpress Mst-1 in treated cells. The levels of autophagosomes and autophagolysosomes in cardiomyocytes undergoing HR and in Mst-1 overexpression+HR group were lower than those of control group, while autophagosomes and autophagolysosomes in cardiomyocytes of Mst-1 knockdown+HR group was significantly higher than in the HR group (all P<0.05). The TUNEL results showed that the proportion of TUNEL positive cells was significantly increased in the HR group and Mst-1 overexpression+HR group than in the control group, while the proportion of TUNEL positive cells was significantly decreased in the Mst-1 knockdown group+HR group as compared to the HR group (all P<0.05). Western blot results showed that the LC3 Ⅱ/LC3 Ⅰ levels were significantly lower, while the expression levels of P62, cleaved-caspase-9 and cleaved-caspase-3 were significantly higher in the HR group and Mst-1 overexpression+HR group than in control group (all P<0.05). The LC3 Ⅱ/LC3 Ⅰ value was significantly higher, and the expression levels of P62, cleaved-caspase-9 and cleaved-caspase-3 were significantly lower in the Mst-1 knockdown+HR group than in the HR group (P both<0.05). The expression level of P-MCL-1 protein was significantly lower in cardiomyocytes of HR and Mst-1 overexpression+HR group than in control group, and the expression level of P-MCL-1 protein was higher in Mst-1 knockdown+HR group than in HR group (P both<0.05). The recovery experiment showed that inhibiting MCL-1 in cells can block the regulatory effect of Mst-1 siRNA on cell autophagy and apoptosis. Conclusion: Inhibiting Mst-1 expression in cardiomyocytes can promote the autophagy of cardiomyocytes induced by hypoxic reoxygenation and reduce the apoptosis of cardiomyocytes via activating McL-1.
Animals ; Apoptosis ; Autophagy ; Hypoxia ; Mice ; Myocytes, Cardiac ; Signal Transduction

Critical ultrasonography(CUS) is different from the traditional diagnostic ultrasound,the examiner and interpreter of the image are critical care medicine physicians.The core content of CUS is to evaluate the pathophysiological changes of organs and systems and etiology changes.With the idea of critical care medicine as the soul,it can integrate the above information and clinical information,bedside real-time diagnosis and titration treatment,and evaluate the therapeutic effect so as to improve the outcome.CUS is a traditional technique which is applied as a new application method.The consensus of experts on critical ultrasonography in China released in 2016 put forward consensus suggestions on the concept,implementation and application of CUS.It should be further emphasized that the accurate and objective assessment and implementation of CUS requires the standardization of ultrasound image acquisition and the need to establish a CUS procedure.At the same time,the standardized training for CUS accepted by critical care medicine physicians requires the application of technical specifications,and the establishment of technical specifications is the basis for the quality control and continuous improvement of CUS.Chinese Critical Ultrasound Study Group and Critical Hemodynamic Therapy Collabration Group,based on the rich experience of clinical practice in critical care and research,combined with the essence of CUS,to learn the traditional ultrasonic essence,established the clinical application technical specifications of CUS,including in five parts:basic view and relevant indicators to obtain in CUS;basic norms for viscera organ assessment and special assessment;standardized processes and systematic inspection programs;examples of CUS applications;CUS training and the application of qualification certification.The establishment of applied technology standard is helpful for standardized training and clinical correct implementation.It is helpful for clinical evaluation and correct guidance treatment,and is also helpful for quality control and continuous improvement of CUS application.

The purpose of this study was to investigate the effect of Huaier on autophagy of human hepatoma SK-HEP-1 cells and the effect of autophagy on the proliferation of SK-HEP-1 cells. CCK-8 assay was used to evaluate the effect of Huaier on the proliferation of SK-HEP-1 cells under different concentrations and different times. Acridine orange staining was used to measure the effect of Huaier on the autolysosome formation in SK-HEP-1 cells. Immunofluorescence assay was applied to examine the effect of Huaier on the expression and distribution of autophagy marker LC3 in SK-HEP-1 cells. In addition， LC3 expression was also checked by immunoblot analysis in the presence of Huaier. At last， the effects of Huaier in combination with autophagy inhibitor bafilomycin A1 on the proliferation of SK-HEP-1 cells was detected by CCK-8 assay. The results showed that Huaier aqueous extract significantly inhibited the proliferation of human hepatoma SK-HEP-1 cells in a dose- and time-dependent manner. Huaier aqueous extract dramatically promoted the formation of autolysosome in SK-HEP-1 cells. Moreover， Huaier markedly increased the number and intensity of intracellular LC3 fluorescent puncta and up-regulated LC3-Ⅱ expression. These data indicated that Huaier evidently activated autophagy of SK-HEP-1 cells. Additionally， autophagy inhibition significantly attenuated the sensitivity of SK-HEP-1 cells to Huaier treatment. Therefore， autophagy activation is involved in the inhibitory effects of Huaier on the proliferation of human hepatoma SK-HEP-1 cells.
Apoptosis ; Autophagy ; Carcinoma, Hepatocellular ; drug therapy ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Complex Mixtures ; pharmacology ; Humans ; Up-Regulation

OBJECTIVETo achieve early diagnosis for inheritable hearing loss and determine carrier rate of deafness causing gene mutations in order to provide information for premarital, prenatal and postnatal genetic counseling.

METHODSA total of 17 000 dried heel blood spots of normal newborns in Chengdu were collected with informed consent obtained from their parents. Genomic DNA was extracted from dried blood spots using Qiagen DNA extraction kits. Microarrays with 9 common mutation loci of 4 deafness-associated genes in Chinese population were used. Nine hot mutations including GJB2 (35delG, 176del16, 235delC and 299delAT), GJB3 (538C> T), SLC26A4 (IVS 7-2A> G, 2168A> G), and mitochondrial DNA 12S rRNA (1555A> G, 1494C> T) were detected by PCR amplification and microarray hybridization. Mutations detected by microarray were verified by Sanger DNA sequencing.

RESULTSOf the 17 000 new-borns, 542 neonates had mutations of the 4 genes. Heterozygous mutations of GJB2, at 235delC, 299delAT, and 176del16 were identified in 254, 55, and 15 newborns, respectively. Two newborns had homozygous mutation of GJB2, 235delC. Heterozygous mutations at 538C> T of GJB3, 2168A> G and IVS 7-2A> G of SLC26A4 were found in 23, 17 and 128 newborns, respectively. For mutation analysis of mitochondrial DNA 12S rRNA, 1494C> T and 1555A> G were homogeneous mutations in 4 and 42 neonates, respectively. In addition, 6 complexity mutations were detected, which demonstrated that one newborn had heterozygous mutations at GJB2 235delC and SLC26A4, IVS7-2A> G, one had heterozygous mutation GJB2 235delC and 12S rRNA homogeneous mutation, 1555 A> G, one heterozygous mutations at GJB2, 299delAT, and GJB3, 538C> T, one at GJB2, 299delAT and 12S rRNA, 1555 A> G, two at GJB2, 299delAT, and SLC26A4, IVS7-2A> G. All mutations as above were confirmed by DNA sequencing.

CONCLUSIONThe total mutation carrier rate of the 4 deafness genes is 3.19% in healthy newborns at Chengdu. Mutations of GJB2 and SLAC26A4 are major ones (86.5% of total). The mutation rate of mitochondrial DNA 12S rRNA is 2.71‰, which may have deafness induced by aminoglycoside antibiotics. Newborn screening for mutation of genes related to hereditary deafness plays an important role in the early detection and proper management for neonatal deafness as well as genetic counseling for premarital, prenatal and postnatal diagnosis.

Asian Continental Ancestry Group ; genetics ; Base Sequence ; China ; Connexin 26 ; Connexins ; genetics ; DNA Mutational Analysis ; DNA, Mitochondrial ; chemistry ; genetics ; Deafness ; diagnosis ; ethnology ; genetics ; Dried Blood Spot Testing ; Genetic Predisposition to Disease ; ethnology ; genetics ; Genetic Testing ; methods ; Humans ; Infant, Newborn ; Membrane Transport Proteins ; genetics ; Microarray Analysis ; methods ; Mutation ; Neonatal Screening ; methods ; RNA, Ribosomal ; genetics

OBJECTIVE: To explore the expression and significance of programmed cell death 5 (PDCD5) and Bcelllymphoma/lewkmia-2(Bcl-2) in sinonasal squamous cell carcinoma (SNSCC). METHOD: Immunohistochemical method and Western Blot method was used to determine the expression of PDCD5 and Bcl-2 in specimen of SNSCC in thirty cases, sinonasal inverted papillomas (SNIP) in thirty-eight cases, and normal nasal mucosa in twenty cases. RESULT: (1) The expression of PDCD5 protein in SNSCC significantly decreased compared with SNIP and normal nasal mucosa. (2) The expression of Bcl-2 protein in SNSCC up-regulated obviously compared with SNIP and normal nasal mucosa. (3) Positive rate of PDCD5 protein and Bcl-2 protein in well, moderate and low differentiatied group is respectively 100.00%, 83.33%, 38.89% and 50.00%, 70.83% and 100.00%, the difference was statistically significant (P < 0.05). (4) In the follow-up cases, the survival rate of the patients with higher expression of PDCD5 protein was higher, but that with lower expression of Bcl-2 protein was higher. CONCLUSION The inactivation of PDCD5 protein and the activation of Bcl-2 protein may play an important role in the development of SNSCC, and there are a positive correlation between PDCD5 and Bcl-2 protein in SNSCC, which may be identified as a new therapeutic target.
Apoptosis Regulatory Proteins ; metabolism ; Carcinoma, Squamous Cell ; metabolism ; mortality ; Head and Neck Neoplasms ; metabolism ; mortality ; Humans ; Neoplasm Proteins ; metabolism ; Papilloma, Inverted ; metabolism ; Paranasal Sinus Neoplasms ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Squamous Cell Carcinoma of Head and Neck ; Survival Rate

OBJECTIVETo explore the optimal treatment selection for treating varicocele (VC) male infertility patients accompanied with oligozoospermia or azoospermia of different Chinese medical syndrome types by comparing the efficacies of integrative medicine.

METHODSOne hundred and twenty male infertility patients with VC accompanied with oligozoospermia or azoospermia were assigned to Chinese medical treatment group (A) and the surgical group (B), each consisting of three Chinese medical syndrome types, i.e., damp-heat stagnation syndrome (DHSS), Shen-deficiency blood stasis syndrome (SDBSS), and blood stasis stagnation syndrome (BSSS), 20 in each group. Corresponding Chinese medical treatment was administered to those in Group A, C, and E, while microscopic ligation of internal vena spermatic was administered to those in Group B, D, and F. The routine analysis of semen, biochemical analysis of seminal plasma, and serum sex hormones (prolactin, testosterone, follicle stimulating hormone, luteinizing hormone, and estradiol) were performed before treatment and by the end of the 24th week after treatment.

RESULTSTotally 18 patients' spouses were pregnant. Of them, 1 in Group A of DHSS (abbreviated as Group A), 3 in Group B of DHSS (abbreviated as Group B), 4 in Group A of SDB-SS (abbreviated as Group C), 5 in Group A of SDBSS (abbreviated as Group D), 1 in Group A of BSSS (abbreviated as Group E), and 4 in Group B of BSSS (abbreviated as Group F). After 24-week treatment, the sperm concentration, class a sperm percentage, class a + b sperm percentage, the motility rate, the seminal plasma of fructose density, and the seminal plasma neutral alpha-glucosidase were more significantly improved in Group B, C, D and F, when compared with the same group before treatment (P <0. 01, P <0. 05). There was no statistical difference in the aforesaid indices between before and after treatment in Group A and Group E (P >0.05). As for the improvement percentage of seminal routine indices, the difference of the seminal plasma of fructose density, and the difference of seminal plasma neutral alpha-glucosidase between before and after treatment in the same Chinese medical syndrome types, better effects were obtained in Group B than in Group A (P <0.01), and better effects were obtained in Group F than in Group E (P <0.01). There was no statistical difference between Group C and D (P >0.05). There was no statistical difference in the 5 items of sex hormones in each group between before and after treatment (P >0.05).

CONCLUSIONSSurgical treatment could effectively improve the semen quality for male infertility VC patients accompanied with oligozoospermia or azoospermia. Of them, Chinese medical treatment could be recommended to those of SDBSS who would not receive surgical treatment.

Adult ; Humans ; Infertility, Male ; diagnosis ; etiology ; therapy ; Integrative Medicine ; Male ; Medicine, Chinese Traditional ; Phytotherapy ; Varicocele ; complications ; diagnosis ; therapy

OBJECTIVETo compare the efficacy of modified D(2) radical total gastrectomy with spleen-preserving and D(2) radical total gastrectomy with splenectomy in patients with gastric cancer located in the upper third, upper and middle third and entire stomach.

METHODSOne hundred and twelve patients with gastric cancer in the upper third, upper and middle third, or entire stomach underwent radical total gastrectomy between January 1989 and December 1994. Modified D(2) total radical gastrectomy with spleen-preserving (spleen-preservation group) was performed in 61 patients, and 51 underwent D(2) total radical gastrectomy with splenectomy (splenectomy group). The differences in clinicopathological characteristics,5-year survival rate, incidence of postoperative complication and hospital stay between the two groups were analyzed retrospectively.

RESULTSThere were no significant differences between the spleen-preservation group and the splenectomy group in gender, age, tumor size, T stage, N stage and TNM stage. The overall 5-year survival rate was 41.0% in the spleen-preservation group and 39.2% in the splenectomy group (P>0.05). The 5-year survival rates of patients with stage I, II, III and IIII were 100%, 66.7%, 27.8% and 17.4% in the spleen-preservation group, respectively, and were 100%, 70.0%, 26.7% and 5.6% in the splenectomy group, respectively (all P>0.05). The incidence of postoperative complication was lower in the spleen-preservation group (11.5% vs 27.5%, P<0.05). The mean hospital stay was longer in the splenectomy group (27.3 d vs 20.3 d, P=0.057).

CONCLUSIONThe efficacy of modified D(2) radical total gastrectomy with spleen-preserving for patients with gastric cancer in the upper third, upper and middle third or entire stomach is similar to that of D(2) radical total gastrectomy with splenectomy, and the spleen-preserving procedure is associated with decreased postoperative complication and improved survival.

Aged ; Female ; Gastrectomy ; Humans ; Lymph Node Excision ; Middle Aged ; Neoplasm Staging ; Prognosis ; Splenectomy ; Stomach Neoplasms ; pathology ; surgery ; Survival Rate ; Treatment Outcome